Construction And Evaluation Of A Novel Braintargeted Liposome Drug Delivery System Mediated By Insulin Receptors

With the aging of human society and the deterioration of the global ecological environment,the incidence of brain diseases such as Alzheimer’s disease,Parkinson’s syndrome,brain tumors,strokes,and brain infections is increasing year by year,therefore,it is of positive significance and value to develop efficient and stable brain drug delivery systems.However,the blood-brain barrier（BBB）is a major obstacle to brain therapy,and a more widely studied strategy is to achieve brain-targeted drug delivery through receptor-ligand interactions based on highly expressed receptor proteins on the BBB.The common receptors mediating brain targeting include insulin receptor,transferrin receptor,LDL receptor,and leptin receptor,which are all membrane protein receptors.The different regions of membrane proteins can be generally divided into extracellular,transmembrane,and intracellular regions.If the extracellular region of membrane proteins is used as the recognition site to build a targeted drug delivery system,there may be two problems:（1）there are natural ligands or endogenous substances that can interact with the extracellular region of membrane proteins in vivo,which may cause competitive interference to the drug delivery system;（2）some extracellular regions of membrane proteins are highly variable,and the ligands designed for the initial extracellular region may not necessarily recognize the mutated extracellular region.Therefore,the above problems are undoubtedly a potential challenge for targeted drug delivery systems constructed by recognizing the extracellular regions of membrane proteins.To effectively avoid the problems of endogenous competitive interference in vivo andthe inability of the extracellular region to be effectively recognized,we explored the feasibility of using the more conserved and stable transmembrane region of membrane proteins as the recognition site to construct a brain-targeted drug delivery system.In this paper,a novel PEG-polymerized brain-targeted liposome drug delivery system（PEG-P-Lip）was constructed using insulin receptor（IR）as the target moiety and an ITP specifically bound to its transmembrane region.Due to the strong hydrophobicity of ITP,it can be spontaneously embedded in the phospholipid layer of liposomes,and this approach can also improve the stability of liposomes without functionalizing peptides by chemical modification.This drug delivery system was used to treat Cryptococcal brain infections using the antifungal drug Amphotericin B（Am B）as a model drug,and its brain-targeted drug delivery and clinical application potential were initially verified.This drug delivery system has the following characteristics:（1）ITP modification can significantly improve the stability of liposomes;（2）based on the recognition of the transmembrane region of IR,it can achieve efficient and stable brain-targeted delivery in vitro and in vivo;（3）it has good immunocompatibility.The specific studies are subdivided as follows.1.Analysis of ITP activity and preparation and characterization of PEG-P-Lip.Based on the ITP obtained from the pre-screening of our group,the surface plasmon resonance（SPR）technique confirmed the high affinity between ITP and IR(K_D=5.08×10^-7M),and molecular docking analysis showed that ITP has a strong binding interaction with the transmembrane region of IR.The results of circular dichroism spectroscopy showed that ITP is anα-helical structure.The effect of ITP modification on the membrane fluidity of liposomes was investigated by using NPD-PE as a membrane probe,and the results showed that ITP modification significantly enhanced the stability of liposomes.Atomic force microscopy（AFM）measurements showed that Young’s modulus after 4%ITP modification of liposomes was about 35 times higher than that of blank liposomes,which significantly improved the elastic modulus of liposomes.The effect of different ratios of ITP modification and DSPE-PEG₂₀₀₀ modification on the active targeting of the formulation was investigated by cell uptake assay,and the screening results showed that the combination of 4%ITP and 5%DSPE-PEG₂₀₀₀modification was the best.The results showed that the PEG-P-Lip was homogeneous in size with an average diameter of about 87.40 nm.The transmission electron microscopy（TEM）and AFM analysis showed that it had a rounded morphology and a distinct liposome structure.Fluorescence resonance energy transfer（FRET）experiments showed that PEG-P-Lip had good serum stability.Further,PEG-P-Lip/Am B encapsulated with the model drug Am B was prepared,and the appearance of the preparation was clarified and translucent with an encapsulation rate of 84.69%,and the in vitro release rate was slightly lower than that of the control group without ITP modification.2.Evaluation of PEG-P-Lip active targeting in vitro and in vivo.This chapter further evaluated the ex vivo active targeting of PEG-P-Lip.Firstly,rat primary brain microvascular endothelial cells（BMEC）were extracted and isolated as model cells,and the targeting mechanism of PEG-P-Lip was investigated after silencing the IR gene（Insr）in BMEC cells by si RNA technology,and the results showed that the uptake of PEG-P-Lip by BMEC cells was significantly reduced after si RNA silencing of Insr,indicating that PEG-P-Lip was actively targeted via IR-mediated active targeting was achieved.The IR extracellular region binding peptide（IEP）was used to saturate the IR extracellular region of b End.3 cells to mimic endogenous interference or the inability of the extracellular region to be effectively recognized.The uptake results showed that the uptake of IEP-modified liposomes（IEP-Lip）was significantly reduced in b End.3 cells,while there was no effect on PEG-P-Lip.It was verified that the site of action of ITP is the IR transmembrane region,and showed that PEG-P-Lip could effectively circumvent the problem of using the extracellular region as the recognition site.In the BBB model,permeation experiments showed that PEG-P-Lip can effectively throughout BBB.In vivo/ex vivo imaging and tissue distribution experiments in animals showed that PEG-P-Lip has significant brain targeting in vivo and that PEG modification can enhance the active effect of only ITP modification mediated active targeting in vivo.The in vivo targeting was again validated in a brain IR conditional knockout mouse model,confirming at the animal level that the targeting mechanism of ITP modifications is IR-mediated.Brain microvascular permeability assays demonstrated that PEG-P-Lip can effectively permeate out of the vasculature and re-enter the brain,contributing to the diffusion of liposomes in the brain parenchyma.RAW 264.7 uptake assay and in vivo pharmacokinetic assay showed that PEG-P-Lip has a long cycling effect with a prolonged half-life.3.Preliminary evaluation of the efficacy and safety of PEG-P-Lip against cryptococcal brain infection.Cryptococcal brain infection was used as a disease model to evaluate the potential of PEG-P-Lip for the treatment of brain diseases.The inhibition effect of PEG-P-Lip/Am B on cryptococci in vitro was evaluated by MIC assay,and the results showed that its MIC value was 2μg/m L,and the in vitro antibacterial ability was slightly lower than that of free Am B and the control group without ITP modification.Based on this model,we investigated the efficacy of PEG-P-Lip/Am B in infected mice.The fungal burden and GMS staining experiments showed that PEG-P-Lip/Am B could effectively reduce the number of Cryptococcus in brain tissue and the degree of infection.The survival rate experiment showed that the survival rate of the PEG-P-Lip/Am B group was still 42%at day 42 after infection,which significantly improved the survival rate and prolonged the survival time of the infected mice.The hemolysis assay,serum biochemical index determination,and H&E staining pathology examination tentatively demonstrated that PEG-P-Lip/Am B had no significant toxic side effects in vitro or in vivo and had a good safety profile.4.Immunocompatibility evaluation of PEG-P-Lip.It has been reported that modification of peptide target moiety on the surface of liposomes improves the immunogenicity in vivo,and given that the drug delivery system constructed herein involves the modification of ITP in the phospholipid layer of liposomes,which reduces the exposure of target moiety to the in vivo environment,this strategy may improve the in vivo immunocompatibility of the formulation.Therefore,the immunocompatibility of PEG-P-Lip was initially examined in this chapter.SDS-PAGE was used to analyze the protein crown adsorbed by PEG-P-Lip,and the results showed that PEG-P-Lip was able to significantly reduce the type and amount of protein adsorbed compared to IEP-Lip.Stimulation by multiple intravenous injections of liposomes,with c（RGDy K）-modified liposomes（RGD-Lip）as a positive control,showed that PEG-P-Lip did not cause changes in body temperature in mice.The results of immunohistochemical and ELISA assays showed that PEG-P-Lip did not cause deposition of immune complexes Ig M and Ig G in tissues,nor did it lead to an increase in serum levels of Ig M and Ig G.ELISA analysis showed that PEG-P-Lip had no significant effect on serum levels of cytokines IL-8,IL-10,and TNF-α,confirming that PEG-P Lip has good in vivo immunocompatibility.In summary,a novel brain-targeted liposome delivery system was constructed using the IR transmembrane region as the target receptor site,and the peptide-targeted moiety was embedded in the phospholipid layer and PEGylated.The system can effectively avoid the problem that the extracellular region of the target protein is competitively bound by endogenous substances or the extracellular region cannot be effectively recognized,has good stability,and shows significant brain targeting in vivo and in vitro.This drug delivery system can effectively treat cryptococcal brain infections and has a greater potential to be used for the treatment of brain diseases,and also has good immunocompatibility.The drug delivery system constructed for the target receptor transmembrane region is simple in structure design,easy to prepare,and has efficient and stable targeting ability,which provides a reference for the subsequent design of other drug delivery systems targeting membrane proteins.